Flight of a Cytidine Deaminase Complex with an Imperfect Transition State Analogue Inhibitor: Mass Spectrometric Evidence for the Presence of a Trapped Water Molecule

Cytidine deaminase (CDA) binds the inhibitor zebularine as its 3,4-hydrate (<i>K</i><sub>d</sub> ∼ 10<sup>–12</sup> M), capturing all but ∼5.6 kcal/mol of the free energy of binding expected of an ideal transition state analogue (<i>K</i><sub>tx</sub> ∼ 10<sup>–16</sup> M). On the basis of its entropic origin, that shortfall was tentatively ascribed to the trapping of a water molecule in the enzyme–inhibitor complex, as had been observed earlier for product uridine [Snider, M. J., and Wolfenden, R. (2001) <i>Biochemistry 40</i>, 11364–11371]. Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) of CDA nebularized in the presence of saturating 5-fluorozebularine reveals peaks corresponding to the masses of E<sub>2</sub>Zn<sub>2</sub>W<sub>2</sub> (dimeric Zn-CDA with two water molecules), E<sub>2</sub>Zn<sub>2</sub>W<sub>2</sub>Fz, and E<sub>2</sub>Zn<sub>2</sub>W<sub>2</sub>Fz<sub>2</sub>, where Fz represents the 3,4-hydrate of 5-fluorozebularine. In the absence of an inhibitor, E<sub>2</sub>Zn<sub>2</sub> is the only dimeric species detected, with no additional water molecules. Experiments conducted in H<sub>2</sub><sup>18</sup>O indicate that the added mass W represents a trapped water molecule rather than an isobaric ammonium ion. This appears to represent the first identification of an enzyme-bound water molecule at a subunit interface (active site) using FTICR-MS. The presence of a 5-fluoro group appears to retard the decomposition of the inhibitory complex kinetically in the vapor phase, as no additional dimeric complexes (other than E<sub>2</sub>Zn<sub>2</sub>) are observed when zebularine is used in place of 5-fluorozebularine. Substrate competition assays show that in solution zebularine is released from CDA (<i>k</i><sub>off</sub> > 0.14 s<sup>–1</sup>) much more rapidly than is 5-fluorozebularine (<i>k</i><sub>off</sub> = 0.014 s<sup>–1</sup>), despite the greater thermodynamic stability of the zebularine complex.